Localized induction of DNA damage in cells

A number of proteins involved in homologous recombination relocalize from a diffuse nuclear distribution to distinct nuclear foci upon treatment of the cells with ionizing radiation. These treatments result in randomly localized DSBs at which homologous recombination proteins accumulate and can then be detected as foci (see Figure: Accumulation of HR proteins at randomly induced sites of DNA damage). To study the coordination of the recruitment of DNA repair factors to damaged DNA, we implemented several methods to induce local damage, such as UV irradiation through micropores, local UV-laser damage induction and multiphoton-damage induction. Probably because the presence of damaged DNA during replication results in an excess number of replication fork arrests and thereby enhanced activation of the DSB repair machinery, these different local damage techniques are also useful to study the coordination of the recruitment and dynamics of DSB repair factors in vivo. To follow the order of events that occur after direct DSB induction and the kinetics, the accumulation of DSB repair proteins to sites of local DSBs at the cellular level we also implemented alpha-particle irradiation (see Figure:  Accumulation of the Rad50 complex at DSBs long an alpha-particle track.)

Accumulation of HR proteins at randomly induced sites of DNA damage.

Accumulation of the Rad50 complex at DSBs long an alpha-particle track.